Characterization and Antimicrobial Activity Assessment of Postbiotic-Loaded Chia Mucilage–Montmorillonite Films Developed for Food Systems

Abstract

This study presents the development and characterization of chia mucilage–montmorillonite (MMT) films enriched with Lactobacillus sakei postbiotics. The films were evaluated mainly for their antimicrobial properties and practical applicability in food systems. The postbiotic contained 21 phenolic compounds and 60 volatile metabolites that exhibited inhibitory activity against Escherichia coli O157:H7 and Listeria monocytogenes. Incorporation of postbiotics and MMT into chia mucilage significantly enhanced antimicrobial performance. Rainbow trout (Oncorhynchus mykiss) fillets were used as a model food system to demonstrate practical applicability, and their shelf life was extended by 9–15 days compared with controls. These findings confirm the potential of postbiotic-loaded chia mucilage–MMT films as promising bioactive packaging materials for food systems, combining natural antimicrobial activity with improved preservation capacity.

1. Introduction

Bioactive packaging has developed rapidly as the food sector seeks sustainable alternatives to conventional plastics, prioritizing materials that both protect products and reduce the environmental burden. Among these, biopolymer films functionalized with natural antimicrobials are especially promising for suppressing foodborne pathogens and delaying spoilage while maintaining quality in refrigerated storage [1]. Recent reviews have provided strong evidence that biopolymer-based functional films, combining polysaccharides with bioactive agents, improve physical integrity and deliver antimicrobial action across diverse food matrices, including fish and poultry [1,2].

Postbiotics—non-viable microbial products and metabolites from probiotic cultures—offer a robust antimicrobial toolkit (organic acids, peptides, phenolics, and volatile compounds) without the viability constraints of live cells. Their stability, safety, and broad-spectrum inhibitory effects make them compelling for integration into food packaging films to curb pathogens and spoilage organisms during cold storage [3,4,5]. The antimicrobial effectiveness of postbiotic-enriched edible films has been increasingly documented, with demonstrated reductions in aerobic plate counts and extended shelf life in chilled products [3].

Chia (Salvia hispanica L.) mucilage is a polysaccharide-rich, film-forming matrix with excellent gelling and oxygen barrier properties and intrinsic antimicrobial potential. Nevertheless, its hydrophilic nature can compromise water resistance and mechanical strength, which has motivated nanofiller reinforcement strategies [6,7,8]. Montmorillonite (MMT), a layered silicate nanoclay, is widely used to strengthen biopolymer films, reduce permeability, and serve as a compatible carrier for antimicrobial actives. MMT-based nanocomposites have shown notable antibacterial performance in food preservation contexts, including AgNP/MMT systems and polyphenol-loaded PVA/MMT films, underscoring the relevance of clay–bioactive synergy in active packaging [9,10,11].

Despite accumulating evidence on postbiotics, chia mucilage matrices, and MMT nanofillers individually, their combined use within a single film to deliver targeted antimicrobial functionality remains underexplored. A rational integration—postbiotic actives (acidic metabolites, phenolics) within a chia mucilage network reinforced by MMT—may produce synergistic antimicrobial performance together with improved barrier and mechanical properties, suitable for sensitive food systems like seafood [3,7,12]. In this study, we developed and characterized Lactobacillus sakei postbiotic-loaded chia mucilage-MMT films, evaluated their ability to inhibit Escherichia coli O157:H7 and Listeria monocytogenes, and demonstrated their practical applicability to improve the microbial and chemical quality of rainbow trout fillets during cold storage, which we used as a model food system.

2. Results and Discussion

2.1. Antimicrobial Potency of Postbiotics

The antibacterial activity of the postbiotics, as well as their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values, was determined against Escherichia coli O157:H7 (ATCC 35150) and Listeria monocytogenes (ATCC 7644). The MIC and MBC values were found to be 25% and 45%, respectively (

Table 1. Minimum inhibition concentration, minimum bactericidal concentration and inhibition zone diameter of Lactobacillus sakei postbiotic (Mean ± SE).

Bacteria	MIC (%)	MBC (%)	Inhibition Zone Diameter (mm)
Escherichia coli O157:H7 (ATCC 35150)	25	45	10.52 ± 0.13
Listeria monocytogenes (ATCC 7644)	25	45	11.67 ± 0.22

). The diameters of the inhibition zones were measured as 10.52 ± 0.13 mm for E. coli O157:H7 and 11.67 ± 0.22 mm for L. monocytogenes.

The number of studies focusing on the characterization of postbiotics produced by lactic acid bacteria (LAB) is still limited. Previous investigations have identified organic acids such as lactic, acetic, formic, caproic, and propionic acids, along with short-chain fatty acids and acetoin, as key constituents of postbiotics [13]. In the present study, 21 distinct compounds were identified, with concentrations higher than those reported in previous research (

Table 2. Content of phenolic and flavonoid components of the Lactobacillus sakei postbiotic (mg/L Mean ± SE).

Component Name	Concentration (mg/L)
Gallic Acid	9.07 ± 1.57
Caftaric	1.50 ± 0.11
Sinapic	1.88 ± 0.05
p-coumaric	2.04 ± 0.01
trans-caffeic acid	30.14 ± 2.23
2,5 Dihydroxy Benzoic	2.89 ± 0.11
Chlorogenic	2.01 ± 0.04
Protokaucic	0.04 ± 0.01
4-Dihydroxy Benzoic	1.05 ± 0.03
Syringic	0.08 ± 0.02
T-Cinnamic Acids	0.33 ± 0.04
Epicatechin	0.12 ± 0.02
Procyanidin B2	0.88 ± 0.10
Catechin	1.63 ± 0.06
Hesperidin	0.17 ± 0.01
Kaempferol	0.63 ± 0.05
Kaempferol-3-Glucoside	0.78 ± 0.00
Quercetin	1.52 ± 0.05
Quercetin-3-Glucoside	1.90 ± 0.02
Luteolin	1.77 ± 0.02
Myricetin	2.43 ± 0.08

and

Table 3. Volatile component profile of the Lactobacillus sakei postbiotic (Peak area percentage ± SE).

Compounds	Area (%)
Aldehydes and Ketones
Acetoin	4.12 ± 0.06
Capronaldehyde	0.12 ± 0.05
Dodecyl aldehyde	0.07 ± 0.02
2 Heptanone	0.02 ± 0.01
2,5-Dimethyl-4-hydroxy-3(2H)-furanone	0.11 ± 0.02
2,5-Dimethyl-2,4-dihydroxy-3(2H)-thiophenone	0.97 ± 0.08
Acids
Acetic acid	22.56 ± 0.47
Butyric acid	0.11 ± 0.02
Isobutyric acid	0.23 ± 0.01
Dodecanoic acid	0.21 ± 0.02
Hexadecanoic acid	1.05 ± 0.12
2-Hydroxy-propanoic acid	35.66 ± 2.13
2-Methyl-propanoic acid	0.06 ± 0.01
9-Octadecenoic acid	0.14 ± 0.02
Alcohols
Heptadecanol	1.03 ± 0.11
3-Methyl-1-butanol	0.05 ± 0.05
1-Dodecanol	0.62 ± 0.03
2-Furanmethanol	0.13 ± 0.02
1,3-propanetriol	0.04 ± 0.05
Esters
Butyl lactate	0.32 ± 0.13
2-Hydroxy-methyl propanoate	0.55 ± 0.03
1,3-Propanediol	0.03 ± 0.03
Hexyl salicylate	0.17 ± 0.05
2-Ethyl-hexyl acetate	0.37 ± 0.11
Linalyl formate	0.12 ± 0.01
Heptyl formate	0.31± 0.02
Methyl-10-Octadecenoate	0.02 ± 0.01
Methyl 10-hydroxy octadecanoate	0.02 ± 0.03
Decyl propanoate	0.14 ± 0.05
Octadecyl acetate	4.21 ± 0.30
Hydrocarbons
Isobutane	0.14 ± 0.03
1-Hexadecene	1.97 ± 0.05
1-Pentadecene	2.04 ± 0.11
Tridecane	0.38 ± 0.02
2,6,10-Trimethyl-dodecane	0.25 ± 0.07
1-Non-nadecene	36.61 ± 0.05
3,3,4-Trimethyl-1-decene	1.12 ± 0.02
Acetylurethane	0.05 ± 0.01
Phenyl and Phenolic Compounds
3-Phenyl-2-propenoic acid	0.36 ± 0.07
Diphenyl-methanone	0.55 ± 0.10
2,4-Di-tert-butyl phenol	16.47 ± 0.31
Benzyl salicylate	0.14 ± 0.05
Dioctyl-1,2-benzenedicarboxylate	0.04 ± 0.02
α-Hydroxy-benzenepropanoic acid	0.63 ± 0.07
Benzylmalonic acid	0.17 ± 0.05
Pyrazine, Piranone and Pyrrole Compounds
2-Methyl pyrazine	0.13 ± 0.05
2-Ethyl-3-methoxy-pyrazine	0.09 ± 0.02
2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one	6.77± 0.08
Pyrrolo [1,2-a]pyrazine-1,4-dione, Hexahydro-3-(2-methylpropyl)	9.65 ± 0.09
Pyrrolo [1,2-a]pyrazine-1,4-dione, Hexahydro-3-(phenylmethyl)	20.67 ± 0.12

). The differences observed between the current findings and earlier reports may be attributed to variations in the composition, concentration, and diversity of bioactive compounds present in the postbiotic preparations.

2.2. Chemical Changes

Figure 1a illustrates the pH changes in rainbow trout fillets coated with the prepared films during 18 days of refrigerated storage at 4 °C. A gradual increase in pH was observed across all treatment groups as storage progressed. However, compared to the control group (Ch), the increase in pH values was significantly lower (p < 0.05) in the groups treated with Ch/1% MMT and Ch/2% MMT.

The initial pH value on day 0 was 6.53, which is consistent with the findings by Giménez et al. [14], although it is slightly higher than the value reported by Kuley et al. [13]. Variations in pH among fresh fish samples are generally attributed to the decomposition of carbonic acid, which leads to gradual alkalinization. While the pH of fresh fish fillets is nearly neutral, the post-mortem breakdown of nitrogenous compounds contributes to a progressive increase in pH, ultimately affecting product quality during storage [15]. By day 18, the pH of the control group (Ch) had risen from 6.53 to 7.80. Samples coated with Ch/1% MMT and Ch/2% MMT exhibited similar trends, with final pH values of 7.20 and 7.00, respectively; however, these differences were not found to be statistically significant (p > 0.05).

These findings are in agreement with those by Souza et al. [16], who reported comparable pH values in meat samples preserved with Ch, Ch + MMT, or Ch + MMT + GEO over a 15-day storage period. TVB-N is one of the most widely used indices of fish quality, and elevated TVB-N values are undesirable as they indicate the presence of nitrogenous compounds resulting from the breakdown of proteins and nucleic acids by proteolytic bacteria [2]. According to European regulation (ECC 95/149), the maximum permissible TVB-N limit is 35 mg N/100 g of fish meat. Since no specific threshold has been established for rainbow trout, a general consensus suggests that the highest acceptable level is 25 mg N/100 g, as reported by Giménez et al. [12]. In the present study, TVB-N values in all samples were 10.02 mg/100 g on day zero (Figure 1b). However, TVB-N levels increased progressively in all groups during storage. The highest concentrations were observed in samples wrapped with Ch, Ch/1% MMT, and Ch/2% MMT films, whereas the lowest levels were detected in the Ch/50% PP and Ch/100% PP groups (p < 0.05).

The increase in TVB-N content in the groups prepared with PP combinations (Ch/1% MMT/50% PP, Ch/1% MMT/100% PP, Ch/2% MMT/50% PP, Ch/2% MMT/100% PP) was relatively small. This reduction may be attributed to the restricted migration of phenolic compounds from postbiotics into fish meat in the presence of MMT, which consequently weakened the protective effect against microbial growth.

However, the incorporation of postbiotics into the chia film matrix effectively reduced the increase in TVB-N during the shelf life of the samples (p < 0.05). The addition of probiotic bacteria to the edible film also demonstrated a beneficial effect on this preservation process. Edible films not only limit the transfer of substances such as water, gases, and oils from the product surface but also inhibit microbial growth. Probiotic bacteria, particularly LAB, are capable of competing with spoilage microorganisms, thereby potentially preventing deterioration and helping to maintain TVB-N levels below acceptable thresholds for human consumption. Moreover, probiotic bacteria may act synergistically with the antibacterial properties of the edible film, resulting in more effective inhibition of the progressive increase in TVB-N [17].

In line with these findings, Karimi et al. [11] reported that edible sodium alginate/whey protein composite coatings containing probiotic bacteria were effective in slowing the rise in TVB-N in rainbow trout fillets compared to both control and pure film treatments. Kuley et al. [13] reported that the application of microencapsulated CFS from L. reuteri, either alone or in combination with propolis extracts, resulted in lower TVB-N content in fish meatballs. The authors attributed these outcomes to the antimicrobial activity of CFS derived from L. reuteri. Our findings are consistent with those reported in these studies.

Lipid oxidation is among the most detrimental processes affecting food quality, leading to the development of off-flavours, stale odours, and undesirable changes in taste. Such alterations are commonly associated with consumer rejection and reduced product acceptability [18,19]. According to Karimi et al. [11], the sensory threshold for unpleasant odour perception in fresh fish corresponds to a TBARS value of 5 mg MDA/kg sample. On day 0, no statistically significant differences in TBA values were observed among trout samples wrapped with the various film formulations (p > 0.05). The initial TBA value was measured as 0.31 mg MDA/kg (Figure 1c).

During storage, TBA levels increased across all groups, likely due to partial dehydration and enhanced oxidation of unsaturated fatty acids. The greatest increase was observed in the control group (Ch), with statistical significance (p < 0.05). According to our findings, samples coated with chia mucilage film (Ch) reached the consumability threshold on day 12 (6.02 mg MDA/kg), whereas the Ch/1% MMT and Ch/2% MMT groups reached this threshold on day 18. No significant difference was detected between the Ch/1% MMT and Ch/2% MMT groups (p > 0.05).

Fluctuations in TBA values were observed in the Ch/50% PP, Ch/100% PP, Ch/1% MMT/50% PP, Ch/1% MMT/100% PP, Ch/2% MMT/50% PP, and Ch/2% MMT/100% PP groups throughout the 18-day storage period. However, none of these groups exceeded the consumability threshold. The differences among bionanocomposite films containing both PP and MMT were not statistically significant (p > 0.05). These fluctuations may be attributed to bacterial degradation of MDA, secondary oxidation reactions that prevent MDA from reacting with the TBA reagent, and tertiary degradation involving interactions between MDA and amino acids, proteins, glucose, or other fish components [20].

In addition to its structural role, montmorillonite (MMT) exhibits antioxidant properties that help protect food from lipid oxidation, thereby extending shelf life. MMT also functions as an effective barrier against moisture and oxygen, which is particularly advantageous for high-fat products [15]. Echeverria et al. [21] reported that protein- and MMT-enriched films effectively protected tuna fillets from lipid autoxidation, with enhanced efficacy when clove oil extract was incorporated. These findings are consistent with our results. Furthermore, the inclusion of probiotic bacteria in the film matrix enhanced antioxidant efficacy. Hybrid probiotic treatments have been shown to provide superior preservation outcomes during storage [15,22].

Recent studies have emphasized the antioxidant potential of probiotic bacteria, particularly LAB strains, which can neutralize reactive oxygen species (ROS) such as peroxide radicals, superoxide anions, and hydroxyl radicals. Several investigations have demonstrated that the direct addition of probiotic strains to processed meats reduces lipid oxidation compared with untreated controls [20].

2.3. Microbiological Changes

In the present study, the initial total viable count (TVC) of rainbow trout fillets was measured at 1.66 log CFU/g (Figure 2a). TVC increased gradually in all treatment groups during refrigerated storage, with the highest increase observed in samples coated with chia mucilage film (Ch) (p < 0.05). According to the International Commission on Microbiological Specifications for Foods [23], the maximum acceptable microbial load for raw freshwater fish is 7 log CFU/g. The Ch group exceeded this threshold on day 12, reaching 7.32 log CFU/g.

In contrast, samples coated with Ch/1% MMT and Ch/2% MMT films surpassed the spoilage limit on day 15, reaching 7.65 and 7.80 log CFU/g, respectively. The lowest TVC values were recorded in the Ch + PP film groups (p < 0.05). Notably, TVC remained below the consumability threshold even after 18 days of storage in the Ch + MMT + PP groups. These findings suggest that antibacterial efficacy increased with higher PP concentrations, while MMT contributed partially to the observed effect.

Kuley et al. [13] reported that microencapsulated Lactobacillus plantarum CFS combined with propolis extract effectively inhibited microbial growth in food systems. Similarly, Jo et al. [17] demonstrated that treatment of ribbonfish fillets with L. plantarum SKD4 and Pediococcus stilesii SKD11 CFS reduced TVC and extended shelf life. Several other studies have also confirmed the antimicrobial activity of lactic acid bacteria against aerobic plate counts (APC), primarily due to the production of organic acids and bacteriocin-like compounds [24].

Psychrotrophic bacteria (PB), one of the primary spoilage agents in refrigerated seafood, increased markedly in all groups during storage (p < 0.05) (Figure 2b). The initial PB count in the Ch group was approximately 1.05 log CFU/g, rising to 7.18 log CFU/g by day 12. In contrast, PB levels remained below the acceptable limit (~7 log CFU/g) throughout the storage period in all PP-supplemented film groups. In the Ch + MMT groups, the spoilage threshold was exceeded on day 15.

Wang et al. [25] demonstrated that postbiotics effectively suppressed spoilage bacteria, particularly psychrotrophs, in liquid egg systems. Similarly, Moradi et al. [26] reported a significant reduction in psychrotroph counts in minced meat treated with CFS derived from L. acidophilus and L. salivarius. These findings are consistent with our results.

Yeast and mould counts were highest in the Ch group and lowest in the Ch/1% MMT/50% PP, Ch/1% MMT/100% PP, Ch/2% MMT/50% PP, and Ch/2% MMT/100% PP groups (Figure 2c). The incorporation of postbiotics and MMT into the chia mucilage film matrix significantly reduced fungal growth during storage (p < 0.05), with postbiotic addition showing a more pronounced effect (p < 0.05). Ceylan et al. [27] similarly reported that chitosan-based nanofiber coatings inhibited yeast and mould growth in fish fillets. The antifungal properties of postbiotics have also been validated in other studies [13,20]. Pseudomonas spp. increased gradually during cold storage (p < 0.05) (Figure 2d). These findings are consistent with previous studies [26]. L. monocytogenes, E. coli O157:H7, and Staphylococcus aureus counts remained <1 log CFU/g in all groups during storage. For this reason, the results were not included in the table. These outcomes are in parallel with the findings by İncili et al. [20].

2.4. Color Analysis

Food colour is a critical factor influencing consumer purchasing decisions. Changes in L* (lightness), a* (redness), and b* (yellowness) values during refrigerated storage are presented in

Table 4. Colour index of rainbow trout fillets wrapped with produced films during storage.

Films	L*	a*	b*	ΔE
Ch 0. days	72.12 ± 0.21	−0.18 ± 0.04	19.22 ± 0.14	4.14 ± 0.55
18. days	60.05 ± 0.12	0.25 ± 0.12	12.20 ± 0.03	4.42 ± 0.06
Ch/%1 MMT 0. days	68.23 ± 0.09	−0.43 ± 0.12	17.51 ± 0.10	3.90 ± 0.12
18. days	54.12 ± 0.14	0.11 ± 0.05	13.55 ± 0.07	4.02 ± 0.13
Ch/%2 MMT 0. days	66.25 ± 0.12	−0.49 ± 0.07	15.10 ± 0.06	3.95 ± 0.04
18. days	50.12 ± 0.17	0.10 ± 0.02	12.36 ± 0.05	4.47 ± 0.11
Ch/%50 PP 0. days	73.66 ± 0.12	−0.22 ± 0.03	22.26 ± 0.14	4.96 ± 0.05
18. days	68.47 ± 0.15	2.45 ± 0.14	20.01 ± 0.11	5.13 ± 0.08
Ch/%100 PP 0. days	73.92 ± 0.52	−0.28 ± 0.02	25.76 ± 0.08	4.41 ± 0.08
18. days	68.90 ± 0.14	2.58 ± 0.05	24.03 ± 0.03	5.29 ± 0.06
Ch/%1MMT/%50 PP 0. days	71.03 ± 0.21	−0.33 ± 0.07	17.45 ± 0.07	4.32 ± 0.09
18. days	65.15 ± 0.36	1.05 ± 0.03	15.29 ± 0.12	3.88 ± 0.11
Ch/%1MMT/%100 PP 0. days	72.20 ± 0.11	−0.25 ± 0.01	17.90 ± 0.15	4.36 ± 0.09
18. days	67.98 ± 0.08	1.23 ± 0.16	16.13 ± 0.08	5.16 ± 0.11
Ch/%2MMT/%50 PP 0. days	70.05 ± 0.15	−0.43 ± 0.02	15.66 ± 0.05	4.44 ± 0.15
18. days	65.55 ± 0.15	0.88 ± 0.08	13.14 ± 0.07	5.26 ± 0.10
Ch/%2MMT/%100 PP 0. days	71.26 ± 0.04	−0.56 ± 0.03	15.20 ± 0.10	4.30 ± 0.08
18. days	67.70 ± 0.12	0.93 ± 0.17	13.66 ± 0.08	5.68 ± 0.11

. The initial L* values ranged from 73.92 to 66.25 across the treatment groups and decreased significantly in all samples by the end of the 18-day storage period (p < 0.05). Among the treatments, films containing MMT exhibited the lowest L* values on day 18 (p < 0.05). The decline in L* values during storage is primarily attributed to alterations in light absorption and scattering, resulting from protein degradation in fish muscle due to microbial activity [28].

Jo et al. [17] reported that immersion in cell-free supernatant (CFS) did not significantly affect the colour of ribbonfish fillets during 120 h of storage at 4 °C. In contrast, Arrioja-Bretón et al. [24] observed significant changes in L*, a*, and b* values of beef samples following CFS treatment. In their study, beef samples were immersed in a CFS-containing marinade for 14 h, with the marinade pH measured at 3.8. By comparison, the lowest pH value recorded in our study was 3.96 (Ch group), with a contact time limited to 3 min.

These discrepancies may be attributed to differences in postbiotic pH levels, application methods, contact duration, and the type of meat used. Such factors can influence protein structure, pigment stability, and ultimately colour retention during storage.

2.5. Determination of Migration of MMT Minerals into Fresh Rainbow Trout

Comprehensive research on the migration of montmorillonite (MMT) from nanocomposite films into food matrices remains scarce. Prior to the industrial-scale application and widespread use of MMT in food packaging, it is essential to conduct migration assessments and in vitro cell culture studies to ensure its safety for both human health and the environment [29]. The U.S. Food and Drug Administration (FDA) has recently approved the use of MMT organoclay as a food contact material under FCN 1410 [30]. Connolly et al. [31] investigated the migration of nanoclays using food simulants and developed nanocomposite packaging materials composed of polylactic acid (PLA) and organoclays. Their study, conducted over 10 days at 40 °C, revealed a maximum migration level of 0.88 ± 0.44 mg/dm², well below the European Union’s regulatory limit of 10 mg/dm². Furthermore, the findings indicated that organoclay migration was minimal and did not exert any cytotoxic effects on human epithelial cells [31,32].

In the present study, no statistically significant increase in iron (Fe) content was observed in rainbow trout fillets after 15 days of contact with MMT-containing films at varying concentrations (p > 0.05) (

Table 5. Iron (Fe), magnesium (Mg) and silicon (Si) concentrations (mg/100 g) of trout packed with films containing montmorillonite clay nanoparticles (1%, 2%).

Films	Fe	Mg	Si
Oncorhynchus mykiss meat	3.00 ± 0.12 c	0.32 ± 0.01 c	0.00 ± 0.00 c
Ch/%1 MMT	3.54 ± 0.20 b	0.88 ± 0.01 b	0.02 ± 0.00 b
Ch/%2 MMT	3.91 ± 0.14 a	1.35 ± 0.01 a	0.03 ± 0.00 a
Ch/%1MMT/%50 PP	3.52 ± 0.21 b	0.86 ± 0.01 b	0.01 ± 0.01 b
Ch/%1MMT/%100 PP	3.52 ± 0.17 b	0.86 ± 0.02 b	0.02 ± 0.01 b
Ch/%2MMT/%50 PP	3.90 ± 0.08 a	1.32 ± 0.02 a	0.03 ± 0.00 a
Ch/%2MMT/%100 PP	3.89 ± 0.12 a	1.31 ± 0.02 a	0.03 ± 0.00 a

Values within each column with the different lowercase are significantly different (p < 0.05).

). However, magnesium (Mg) and silicon (Si) levels were significantly elevated compared with fresh trout samples (p < 0.05). According to European Commission Directive 2008/100/EC, the recommended daily intake of magnesium is 375 mg, and the detected migration levels remained well below the EU’s safety threshold of 10 mg/dm².

The increase in Si content can be attributed to the elemental composition of clay nanoparticles, which predominantly consist of silicon. Dias et al. [33] reported similar findings, noting elevated Mg and Si concentrations in salmon fillets packaged with MMT-enriched films compared to untreated samples (p < 0.05). Conversely, another study found no increase in Mg levels in vegetables wrapped with MMT-starch films, although Si levels were elevated [34]. The authors suggested that such variations may be due to the intrinsic properties of the food matrix, including moisture content, pH, and surface composition.

3. Conclusions

This study demonstrated that chia mucilage–MMT films enriched with L. sakei postbiotics possess strong antimicrobial activity and enhanced physicochemical properties. Application to rainbow trout fillets extended shelf life by 9–15 days, confirming their effectiveness in inhibiting microbial growth and maintaining chemical freshness. The integration of postbiotics and nanoclays into biopolymer films represents a promising strategy for developing sustainable, bioactive packaging materials applicable to diverse food systems. Future research should focus on migration control, industrial scalability, and consumer safety to facilitate adoption in commercial seafood packaging.

4. Materials and Methods

4.1. Materials

Chia seeds were purchased from local markets in Elazığ, Türkiye. The Lactobacillus sakei strain (Bactoferm™ B-FM) was obtained from Chr. Hansen GmbH (Wiesbaden, Germany). Montmorillonite (MMT) and all other analytical-grade chemicals were supplied by Sigma-Aldrich (St. Louis, MO, USA).

4.2. Preparation of Postbiotics

Postbiotics were produced using L. sakei (Bactoferm™ B-FM). The culture was incubated in MRS broth under anaerobic conditions at 37 °C for 48 h. After incubation, cells were removed by centrifugation (4200× g, 10 min, 4 °C), and the supernatant was filtered through 0.45 μm membrane filters (MF-Millipore, Merck, Darmstadt, Germany). The filtrate was stored at 4 °C until further use. Antimicrobial and antioxidant activities were subsequently evaluated [26].

4.3. Characterization of Postbiotics

Antibacterial activity was determined using the disc diffusion method [35]. Phenolic compounds were identified and quantified by HPLC according to Kocabey et al. [36]. Volatile compounds were analyzed by HS-SPME-GC-MS (Shimadzu GC-2010/QP-2010 MS, Kyoto, Japan) equipped with a DB-5MS column.

4.4. Extraction of Chia Seed Mucilage

Chia seed mucilage was extracted following the protocol described by Emir Çoban and Jamshidi [37], with slight modifications to optimize yield and purity. Initially, chia seeds were combined with distilled water at a seed-to-water ratio of 1:20 and stirred continuously with a magnetic stirrer (Daihan SMHS, Wonju, Republic of Korea) at 25 °C for 2 h to facilitate mucilage release. The resulting suspension was subjected to centrifugation at 11,600× g for 30 min using a refrigerated centrifuge (Nüve NF 1200 R, Nüve Industrial and Commercial Inc., Sincan, Turkey) to separate the soluble fraction from seed residues. The supernatant was then filtered through a double-layered cheesecloth to remove insoluble particles and obtain a clear mucilage solution. The collected mucilage was subsequently dried in a hot-air oven (Nüve Industrial and Commercial Inc., Sincan, Turkey) at 50 °C for approximately 6 h until a constant weight was achieved. The dried material was finely ground using a laboratory mill (IKA^®-Werke GmbH & Co. KG, Breisgau, Germany), sieved to ensure uniform particle size, and stored in airtight containers under dry conditions for subsequent experimental applications.

4.5. Preparation of Film Solutions

Chia mucilage powder was dissolved in distilled water (0.5%, w/v) at 75 °C. MMT was added at 1% and 2% (w/w relative to mucilage), and homogenized (Ultra-Turrax T25, IKA^®, Staufen im Breisgau, Germany, 15,000 rpm, 5 min). Postbiotics were incorporated at 50% and 100% (v/v). Glycerol (0.6%, w/v) was added as a plasticizer. Film-forming solutions (30 mL) were cast in Petri dishes and dried at 35 °C for 24 h. Nine formulations were prepared (

Table 6. Postbiotic loaded chia mucilage-montmorillonite film applications.

Experimental Groups	Applications
Ch	Film made with pure chia mucilage
Ch/%1 MMT	Film made with chia and 1% MMT
Ch/%2 MMT	Film made with chia and 2% MMT
Ch/%50 PP	Film made with chia and 50% postbiotic
Ch/%100 PP	Film made with chia and 100% postbiotic
Ch/%1MMT/%50 PP	Film made with chia, 1% MMT and 50% postbiotic
Ch/%1MMT/%100 PP	Film made with chia, 1% MMT and 100% postbiotic
Ch/%2MMT/%50 PP	Film made with chia, 2% MMT and 50% postbiotic
Ch/%2MMT/%100 PP	Film made with chia, 2% MMT and 100% postbiotic

).

4.6. Antimicrobial Activity of the Films

The antibacterial properties of the films against Escherichia coli O157:H7 (ATCC 35150) and Listeria monocytogenes (ATCC 7644) were evaluated using the disk diffusion assay. The bacterial strains were subcultured twice in tryptic soy (TS) broth and incubated at for 18 h. Prior to testing, the bionanocomposite films were sterilized by ultraviolet irradiation in a laminar flow cabinet for 2 min. Subsequently, 100 µL of a 20 h bacterial suspension adjusted to cfu/mL was spread onto TS agar plates. Wells of 9 mm diameter were prepared using a sterile cork borer, into which the film solutions were introduced. The plates were then incubated at for 24 h, and the diameters of the resulting inhibition zones were measured with a digital caliper [37].

4.7. Application to Rainbow Trout Fillets

Fresh rainbow trout (Oncorhynchus mykiss) were obtained from a local trout farm (Gümüşdoğa A.Ş., Elazığ, Turkey). Fish weighing approximately 400 ± 50 g were immediately transported to the Aquaculture Processing Technology Laboratory under aseptic conditions. Internal organs, skin, and bones were removed, and the fish were filleted. The fillets were rinsed with sterile distilled water, drained on sterile stainless steel wire mesh, and cut into uniform portions weighing 25–30 g.

Each fish portion was individually wrapped with the film formulations described in Table 6 and packaged aerobically in sterile styrofoam boxes (Figure 3). The packaged samples were stored at 4 °C and analyzed every three days throughout the storage period. Evaluations included chemical parameters (pH, total volatile basic nitrogen [TVB-N], and thiobarbituric acid [TBA] values), microbial counts (total viable count, psychrotrophic bacteria, yeast and mould, Pseudomonas spp., Listeria monocytogenes, Escherichia coli O157:H7, and Staphylococcus aureus), and colour measurements.

All analyses were performed in triplicate, and the entire study was conducted with three independent replications.

4.7.1. Chemical Analyses

A digital pH meter (EDT, GP 353, Cole-Parmer Ltd., Watford, UK) was used to determine the pH values of postbiotic chia mucilage solutions and trout fillets during storage, and AOAC [38] procedures were applied. TVB-N determination was conducted using the method provided by [14,39]. TBA analysis was performed according to the method reported by Tarladgis et al. [40].

4.7.2. Microbiological Analyses

For microbiological analyses, 10 g of each sample was aseptically transferred into a sterile stomacher bag containing 90 mL of 0.1% peptone water to obtain a 10⁻¹ dilution. Serial dilutions were then prepared up to 10⁻⁸ as needed.

Total viable count (TVC) and psychrotrophic bacteria were enumerated following the method recommended by USDA-FSIS. Plate Count Agar (Biokar, Plailly, France) was used for both analyses. Inoculated plates for TVC were incubated at 35 ± 2 °C for 48 h, while plates for psychrotrophic bacteria were incubated at 7 ± 1 °C for 7 days. Yeast and mould counts were determined using Dichloran Rose Bengal Chloramphenicol (DRBC) Agar (Biokar, France). The plates were incubated at 25 ± 1 °C for 5 days. Pseudomonas spp. were enumerated using Cephaloridine–Fucidin–Cetrimide (CFC) Agar (Biokar, France), with incubation at 25 °C for 48 h. Listeria monocytogenes and Escherichia coli O157:H7 were detected using Oxford Agar and Sorbitol MacConkey Agar supplemented with cefixime-tellurite (CT), respectively. Staphylococcus aureus was enumerated using Baird-Parker Agar. All media were obtained from Biokar (France), and incubation conditions followed the respective standard protocols [23].

4.7.3. Colour Analysis

A colourimeter was used to measure L*, a* and b* of the samples. The colour characteristics of trout fillets wrapped in the produced films were determined by measuring three different points on the outer surface. Before the analyses. the colorimeter was standardised using standard black and white plates [37].

4.7.4. Migration Analysis of MMT Minerals

To assess the potential migration of montmorillonite (MMT) clay components into food, rainbow trout samples coated with the bioactive films were analyzed at the end of the storage period [33].

4.8. Statistical Analysis

Results are reported as mean ± SD. Data were analyzed using one-way ANOVA in SPSS software (version 26). Duncan’s test was used to determine whether there was a significant difference between means at the 95% confidence level (p < 0.05). All experiments were carried out in three repetitions.